PROJECT SUMMARY Klebsiella pneumoniae is an increasingly prevalent drug resistant nosocomial pathogen. Commonly, K. pneumoniae isolates are carbapenem resistant, which are referred to as carbapenem-resistant Enterobacteriaceae (CRE), and present significantly less therapeutic intervention options. Furthermore, community-acquired infections associated with hypervirulent isolates of K. pneumoniae have emerged that are capable of causing a disseminated disease in healthy individuals. Molecular studies indicate that the main virulence factor of K. pneumoniae is a thick capsular polysaccharide that facilitates immune evasion. Although 79 Klebsiella capsular polysaccharides have been identified, studies indicate that two serotypes, the K1 and K2 serotypes, account for two-thirds of all community-acquired infections, and also cause a significant portion of hospital-acquired infections. We are running out of antibiotics to treat drug resistant K. pneumoniae, and therefore, a prophylactic vaccine would be an alternative method to drastically reduce the burden of K. pneumonia infections globally. Currently there is no licensed vaccine available for individuals at risk of K. pneumoniae infection; indeed, Klebsiella vaccines are not even undergoing clinical trials. Conjugate vaccines, composed of polysaccharides linked to proteins, are ideal vaccines for K. pneumoniae as conjugate vaccines are highly protective and generate immunological memory in all age groups. However, their synthesis is complex, costly, and not conducive for all polysaccharides. To fill this void, VaxNewMo will generate the first conjugate vaccine against two of the most frequently encountered K. pneumoniae serotypes, K1 and K2, using our propriety conjugating enzyme technology. With our platform, we utilize a glycoengineering approach to generate conjugate vaccines in vivo using the lab safe E. coli through a process termed bioconjugation. This glycoengineering strategy eliminates the need for harsh chemicals to covalently link a polysaccharide to a protein. Furthermore, our conjugating enzyme is the first and only enzyme able to transfer K. pneumoniae capsular polysaccharides to an acceptor protein, a feat other conjugating enzymes are unable to perform. The proposed research in this phase I application will focus on (Aim 1) glycoengineering two strains of E. coli for the heterologous expression of the K. pneumoniae K1 and K2 capsular polysaccharides. We will then pair these K1/K2 polysaccharide producing E. coli strains with our proprietary conjugating enzyme technology to develop the first bivalent bioconjugate vaccine against K1 and K2 serotypes of K. pneumoniae. Subsequently (Aim 2), we will demonstrate the immunogenicity and efficacy of our bivalent K. pneumoniae K1/K2 specific bioconjugate vaccine compared to polysaccharide alone as there currently is no vaccine to compare non-inferiority against. Our next step for phase II funding will be to expand the serotype coverage in our bioconjugate vaccine to include the remaining six hypervirulent serotypes as well as demonstrate the safety, immunogenicity, and efficacy of the first bioconjugate vaccine against K. pneumoniae to streamline FDA regulatory approval.